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Neutrophil Gelatinase-AssociatedLipocalin for Acute Kidney Injury DuringAcute Heart Failure HospitalizationsThe AKINESIS Study

Alan S. Maisel, MD,a,b Nicholas Wettersten, MD,b Dirk J. van Veldhuisen, MD,c Christian Mueller, MD,d

Gerasimos Filippatos, MD,e Richard Nowak, MD,f Christopher Hogan, MD,g Michael C. Kontos, MD,h

Chad M. Cannon, MD,i Gerhard A. Müller, MD, PHD,j Robert Birkhahn, MD,k Paul Clopton, MS,a Pam Taub, MD,b

Gary M. Vilke, MD,l Kenneth McDonald, MD,m,n Niall Mahon, MD,n,o Julio Nuñez, MD,p Carlo Briguori, MD, PHD,q

Claudio Passino, MD,r Patrick T. Murray, MDn

ABSTRACT

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BACKGROUND Worsening renal function (WRF) often occurs during acute heart failure (AHF) and can portend adverse

outcomes; therefore, early identification may help mitigate risk. Neutrophil gelatinase-associated lipocalin (NGAL) is a

novel renal biomarker that may predict WRF in certain disorders, but its value in AHF is unknown.

OBJECTIVES This study sought to determine whether NGAL is superior to creatinine for prediction and/or prognosis of

WRF in hospitalized patients with AHF treated with intravenous diuretic agents.

METHODS This was a multicenter, prospective cohort study enrolling patients presenting with AHF requiring intrave-

nous diuretic agents. The primary outcome was whether plasma NGAL could predict the development of WRF, defined as

a sustained increase in plasma creatinine of 0.5 mg/dl or $50% above first value or initiation of acute renal-replacement

therapy, within the first 5 days of hospitalization. The main secondary outcome was in-hospital adverse events.

RESULTS We enrolled 927 subjects (mean age, 68.5 years; 62% men). The primary outcome occurred in 72 subjects

(7.8%). Peak NGAL was more predictive than the first NGAL, but neither added significant diagnostic utility over the

first creatinine (areas under the curve: 0.656, 0.647, and 0.652, respectively). There were 235 adverse events in 144

subjects. The first NGAL was a better predictor than peak NGAL, but similar to the first creatinine (areas under the curve:

0.691, 0.653, and 0.686, respectively). In a post hoc analysis of subjects with an estimated glomerular filtration

rate <60 ml/min/1.73 m2, a first NGAL <150 ng/ml indicated a low likelihood of adverse events.

CONCLUSIONS Plasma NGAL was not superior to creatinine for the prediction of WRF or adverse in-hospital outcomes.

The use of plasma NGAL to diagnose acute kidney injury in AHF cannot be recommended at this time. (Acute Kidney

Injury Neutrophil Gelatinase-Associated Lipocalin [N-GAL] Evaluation of Symptomatic Heart Failure Study [AKINESIS];

NCT01291836) (J Am Coll Cardiol 2016;68:1420–31) © 2016 by the American College of Cardiology Foundation.

m the aDivision of Cardiovascular Medicine, Veterans Affairs Medical Center, San Diego, La Jolla, California; bDivision of

rdiovascular Medicine, University of California, San Diego, La Jolla, California; cDepartment of Cardiology, University Medical

nter Groningen, University of Groningen, Groningen, the Netherlands; dDepartment of Cardiology, University Hospital Basel,

sel, Switzerland; eDepartment of Cardiology, Athens University Hospital Attikon, University of Athens, Athens, Greece; fDe-

rtment of Emergency Medicine, Henry Ford Hospital System, Detroit, Michigan; gDivision of Emergency Medicine and Acute

re Surgical Services, VCUMedical Center, Virginia Commonwealth University, Richmond, Virginia; hDivision of Cardiology, VCU

dical Center, Virginia Commonwealth University, Richmond, Virginia; iDepartment of Emergency Medicine, University of

nsas Hospital, Kansas City, Kansas; jDepartment of Nephrology and Rheumatology, University Medical Center Göttingen,

iversity of Göttingen, Göttingen, Germany; kDepartment of Emergency Medicine, New York Methodist, Brooklyn, New York;

partement of Emergency Medicine, University of California, San Diego, La Jolla, California; mDepartment of Cardiology, St.

cent’s University Hospital, Dublin, Ireland; nDepartment of Nephrology, Mater Misericordaie University Hospital, University

llege Dublin, Dublin, Ireland; oDepartment of Cardiology, Mater Misericordaie University Hospital, Dublin, Ireland; pDepartment

AB BR E V I A T I O N S

AND ACRONYM S

AHF = acute heart failure

AKI = acute kidney injury

AUC = area under the curve

BUN = blood urea nitrogen

CI = confidence interval

eGFR = estimated glomerular

filtration rate

HF = heart failure

IV = intravenous

NGAL = neutrophil gelatinase-

associated lipocalin

RRT = renal-replacement

therapy

J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6 Maisel et al.S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1 Plasma NGAL for AKI in AHF

1421

H eart failure (HF) is a major global healthproblem, with more than 23 million peopleafflicted worldwide (1). Kidney dysfunction

is common among patients hospitalized with acuteHF (AHF) and portends a worse prognosis (2–4). Astrong interaction is recognized between the heartand kidney, and worsening renal function (WRF)can occur with treatment of AHF with adverse out-comes (5,6). Creatinine is currently the standardbiomarker for renal function; however, it has adelayed increase after kidney injury. Furthermore,WRF in AHF, reflected by a rise in creatinine, maynot reflect acute kidney injury (AKI) and may not beprognostic in all patients (7). Novel biomarkers areneeded for earlier detection of WRF that is associatedwith adverse outcomes (8).

SEE PAGE 1432

WRF = worsening renal

function

Neutrophil gelatinase-associated lipocalin (NGAL)is a small molecule of the lipocalin family of proteins.Found on the brush-border of renal tubular cells,NGAL increases during the acute phase of toxic andischemic kidney injury (9). Plasma NGAL has predic-tive value for AKI in multiple conditions, includingcardiac surgery, contrast-induced nephropathy, andcritical illness (10–12). It is also elevated in patientswith chronic HF and is associated with adverse out-comes, but its value in patients with AHF is not wellestablished (13,14). Single-center studies have sug-gested that NGAL predicts WRF in AHF; however, it isunknown if this can be reproduced on a larger scale,or if NGAL is specifically useful with intravenous (IV)diuretic therapy (15–17).

AKINESIS (Acute Kidney Injury N-gal Evaluation ofSymptomatic heart faIlure Study) is an international,multicenter, prospective cohort study enrolling pa-tients presenting with AHF. A primary goal was toexamine plasma NGAL’s ability to predict WRF orneed for renal-replacement therapy (RRT) in patientswith AHF treated with diuretic agents. A secondary

of Cardiology, Hospital Clínico Universitario Valencia, University of Valencia

Mediterranea, Naples, Italy; and the rDepartment of Cardiology and Cardiov

Pisa, Italy. Abbott Laboratories (Chicago, Illinois) and Alere (San Diego, Califo

design of the study, data management, and study oversight. The sponsors

paper or in preparation, review, or approval of the paper. Dr. Maisel previou

and Alere; and previously served as a consultant and speaker for Abbott Labo

grants and speakers/consulting honoraria from several diagnostic companie

and Provencio. Dr. Cannon has received institutional research support from

advisory board of Protagen Protein Service, PPS, Germany; and has a

Dr. Birkhahn has received a grant from Alere. Dr. Murray has received resear

has served as consultant for Abbott Laboratories. All other authors have rep

contents of this paper to disclose. Inder Anand, MD, DPhil (Oxon), served a

Manuscript received April 29, 2016; revised manuscript received June 19, 20

goal was to assess prognostic ability for in-hospital adverse outcomes.

METHODS

STUDY DESIGN. AKINESIS was an interna-tional, multicenter, prospective cohort studyenrolling patients presenting to the emer-gency department or hospital with signs andsymptoms of AHF, with planned admissionand treatment with diuretic agents. AbbottLaboratories and Alere, Inc., jointly spon-sored the study. Subjects were enrolled at 16sites, 7 in the United States and 9 in Europe,and followed to discharge (NCT01291836).

The principal investigators and sponsorsdesigned and oversaw the trial. Each center’sinstitutional review board approved the

study. All participants provided written informedconsent. Data were collated at a core data manage-ment facility. The principal investigators had full ac-cess to the database. An independent biostatisticianperformed statistical analysis. The initial manuscriptwas written by the first 2 authors, and reviewed andedited by all authors. All authors vouch for the ac-curacy of the reported findings.

PARTICIPANTS. Subjects at least 18 years of age,presenting to the emergency department or hospitalwith AHF, were screened for inclusion and enrolled asearly as possible. Subjects had to have 1 or more signsor symptoms of HF, including dyspnea on exertion,rales or crackles, galloping heart rhythm, jugularvenous distention, orthopnea, paroxysmal nocturnaldyspnea, using more than 2 pillows to sleep, fatigue,edema, frequent coughing, a cough that producesmucous or blood-tinged sputum, or a dry cough whenlying flat. In addition, subjects must have received orplanned treatment with IV diuretic agents. Subjectshad to be able to comply with all aspects of the pro-tocol and give consent.

, Valencia, Spain; qDepartment of Cardiology, Clinica

ascular Medicine, Fondazione Gabriele Monasterio,

rnia) funded the study. The sponsors assisted in the

did not participate in the analysis presented in this

sly received grant funding from Abbott Laboratories

ratories and Alere. Dr. Mueller has received research

s. Dr. Kontos is a consultant for Roche, AstraZeneca,

Abbott and Alere. Dr. Müller is a member of the

financial relationship with Celgene and Novartis.

ch funding from Abbott Laboratories and Alere; and

orted that they have no relationships relevant to the

s Guest Editor for this paper.

16, accepted June 28, 2016.

Maisel et al. J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6

Plasma NGAL for AKI in AHF S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1

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Subjects were excluded if: 1) symptoms consistentwith acute coronary syndrome were the chief cause ofthe current AHF episode; 2) they were already ondialysis before enrollment or if dialysis initiationwas planned during the current hospitalization;3) they had a heart, lung, kidney, or liver transplant;4) they had participated in a drug treatmentstudy within the past 30 days or had already enrolledin this study; or 5) they were pregnant or belonging toan institutional review board–determined vulnerablepopulation.

ENDPOINTS. The primary outcome was whetherplasma NGAL predicted the development of eitherWRF, reflected by a sustained increase in creatinineof $0.5 mg/dl (44.2 mmol/l) or $50% of the firstcreatinine value obtained, or the initiation of acuteRRT within 5 days of being hospitalized. A sustainedincrease was defined as a creatinine value meetingcriteria on at least 2 consecutive days. Acute RRTmodalities included dialysis, ultrafiltration, andhemofiltration. The predictive ability of NGAL foreach endpoint of WRF and RRT was also tested.

The major secondary endpoint was the ability ofNGAL to predict adverse in-hospital outcomes. Theseincluded severe WRF (a sustained increase $100% ofthe first creatinine value); initiation of RRT; death;nephrology consultation; and admission to theintensive care unit with need for mechanical venti-lation, inotropic/vasopressor support, or both.

Pre-specified secondary endpoints includedwhether an elevated NGAL on admission predictedwhich subjects with an admission estimated glomer-ular filtration rate (eGFR) <60 ml/min/1.73 m2 wouldhave a greater decrease in eGFR at discharge. Pre-specified alternative definitions of WRF includeda sustained increase in creatinine $0.3 mg/dl(26.5 mmol/l) or $50% of the first creatinine, and asustained increase in creatinine $50% of the firstcreatinine, both during the first 5 days of hospitali-zation. Although the latter definition is similar to RI-FLE (Risk, Injury, Failure, Loss of kidney function,and End-stage kidney disease) criteria, the KDIGO(Kidney Disease: Improving Global Outcomes) criteriafor AKI, which are similar to AKIN (Acute KidneyInjury Network) criteria, were published during studyenrollment. This definition did not use a sustainedincrease in creatinine; thus, post hoc analyses withoutsustained increases were performed. These included anonsustained increase of $0.5 mg/dl or $50% ofthe first creatinine and a nonsustained increase increatinine of $0.3 mg/dl or $50% of the first creati-nine, both during the first 5 days of hospitalization,and any nonsustained increase of $0.3 mg/dl or

$50% of the first creatinine at any time during thehospitalization.

Another post hoc analysis, prompted by an evolu-tion in published reports on the evaluation of novelrenal biomarkers during the study, tested the pre-dictive value of NGAL stratified by admission eGFRand first NGAL for the combined endpoint of in-hospital adverse outcomes (8). eGFR was stratifiedat 60 ml/min/1.73 m2. NGAL was assessed using 2cutpoints: the 80% sensitivity cutpoint, on the basisof our analysis, and at 150 ng/dl, on the basis of aprior study (11).

The primary predictive metric was the peak NGALvalue. The predictive value of the first NGAL, firstcreatinine, and the combination of the first creatinineand first NGAL were also analyzed. Peak NGAL wasdefined as the highest value obtained at least 6 hbefore the event, if the event time was available, or atleast a day before the event, if only the event date wasavailable. If the event occurred on the first day, or theonly available NGAL value was within 6 h of theevent, this was used. If an NGAL value was onlyavailable after the event occurred, this was not used.This peak value for subjects with an outcome wascompared with the peak value from all collected sam-ples in subjects without the outcome. The first NGALwas the value obtained only at the first collection time.The first creatinine was the first value obtained.

DATA COLLECTION. After subjects were screenedand gave written consent, demographics, vital signs,HF signs and symptoms on presentation, medica-tions, and past medical history were collected. Lab-oratory samples were collected as per standard of caredetermined by the treating physician, and wereanalyzed at the clinical laboratories of the treatinghospital. If collected, values recorded on admissionand discharge included white blood cell count, he-moglobin, hematocrit, sodium, potassium, blood ureanitrogen (BUN), creatinine, alanine aminotransferase,aspartate aminotransferase, troponin, and natriureticpeptides.

During the hospitalization, sodium, potassium,BUN, and creatinine values were recorded for at leastthe first 7 days of hospitalization. These were ob-tained at the discretion of the treating physician. In-hospital adverse outcomes listed previously wererecorded.

NGAL ASSESSMENT. Specimens for NGAL assess-ment were collected at 6 time points if the subjectremained hospitalized during the collection period.The first specimen was collected the day of enroll-ment, within 2 h of the first IV diuretic dose.The second specimen was collected 2 to 6 h later.

J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6 Maisel et al.S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1 Plasma NGAL for AKI in AHF

1423

The third, fourth, and fifth specimens were collectedon hospital day 1, 2, and 3, respectively. The sixthspecimen was collected on the day of discharge oranticipated discharge. Compliance with samplecollection was high, except for the discharge sample,with 98.3%, 92.0%, 94.7%, 96.7%, 94.5%, and 65.4%of samples collected at the listed time points. PlasmaNGAL specimens were frozen and shipped to the corelaboratory for analysis with the Alere Triage plasmaNGAL assay.

STATISTICAL ANALYSES. Assuming an event rate of9% and an area under the curve (AUC) of 0.70 for NGALfor the primary outcome, it was estimated that 60primary events had to occur to have 80% power todetect a difference of 0.10 in AUC. With a 20% marginof error, anticipated enrollment was 800 subjects;however, as enrollment neared 800, the event rate waslower than anticipated. It was decided to continueenrollment until at least 60 events had occurred,resulting in 930 subjects enrolled. The institutionalreview boards were notified of the planned increaseand no site exceeded its enrollment limit.

Baseline variables are presented as means andstandard deviations for normally distributed contin-uous variables, medians with interquartile rangesfor nonnormalized variables, and percentages forcategorical variables. When the status of a comor-bidity was unknown, it was assumed to not bepresent.

Baseline characteristics between subjects with andwithout the primary outcome were compared usingthe Student t test, chi-square test, and Mann-WhitneyU test, as appropriate. To explore the independentpredictive value of NGAL and potential influence ofcovariates on the primary outcome, univariable lo-gistic regression analysis was used. Variables with ap value <0.10 were retained for forward, stepwise,multivariate logistic regression analyses, and thosewith a p value <0.05 were retained for a multivariatemodel. The log-transformed values of peak and firstNGAL were added separately to the model, and the Cstatistic was calculated with and without NGAL.When NGAL and creatinine were assessed in combi-nation, logistic regression was used and predictorswere log-transformed. Receiver operating character-istic curves were generated to determine the AUCwith 95% confidence intervals (CIs) for peak NGAL,first NGAL, first creatinine, and combined first NGALand first creatinine for the primary and secondaryoutcomes. Cutpoints for 80% sensitivity and 80%specificity were determined. Kaplan-Meier curveswere created for time to events of the primary andcombined secondary endpoint. Spearman correlation

was used to assess change in subjects with eGFR<60 ml/min/1.73 m2 and NGAL values. Subgroupswere compared by chi-square test, and sensitivity,specificity, and positive and negative predictivevalues in the specific subgroups were calculated.A p value <0.05 was considered significant. All sta-tistical calculations were performed on SPSS version19 (SPSS, Inc., Chicago, Illinois).

RESULTS

PATIENT DEMOGRAPHICS. From January 2011through September 2013, a total of 930 subjects wereenrolled. One subject was later found not to meetinclusion criteria, 1 met exclusion criteria, and 1withdrew consent, leaving 927 subjects for analysis.All subjects had follow-up throughout hospitaliza-tion. Subjects were hospitalized for a median of5 days (interquartile range: 6 days).

The mean age of subjects was 68.5 years, with 62%men and 66.9% identified as white (Table 1). Mostsubjects had hypertension (80.7%), a significantportion had coronary artery disease (46.1%) anddiabetes mellitus (43.6%), and almost 26% hadchronic kidney disease. The median admissioncreatinine was 1.19 mg/dl, with a median eGFR of57 ml/min/1.73 m2. The median admission B-typenatriuretic peptide and N-terminal pro-B-type natri-uretic peptide values were 795 pg/dl and 3,446 pg/dl,respectively. Approximately 70% of subjects were ondiuretic agents before admission, 70% were on beta-blockers, and almost 63% were on an angiotensin-converting enzyme inhibitor or angiotensin-receptorblocker.

PRIMARY OUTCOME. Seventy-two subjects (7.8%)had the primary outcome (Figure 1A), with 66 (7.1%)subjects having WRF and 11 (1.2%) requiring RRT.Subjects with and without the primary outcome weresimilar, except that subjects with the primaryoutcome had a higher prevalence of diabetes andchronic kidney disease (Table 1). The latter was re-flected by significantly higher admission creatinine,lower eGFR, and higher BUN. Hemoglobin was alsosignificantly lower. Troponin T was significantlyhigher in subjects with the primary outcome, whereastroponin I was not different.

A total of 918 subjects had peak NGAL values (9subjects did not have any samples for NGAL analysis),and 911 had first NGAL values (16 lacked a samplefrom the first collection time). Peak values occurred amean of 1.6 � 1.1 days before the primary outcome,with most events occurring 24 h after admission(Figure 1A). Distributions of peak NGAL and first NGAL

TABLE 1 Baseline Characteristics of Study Patients and Comparison of Those With and Without Primary Outcome

N Total Primary Outcome No Primary Outcome

Age, yrs 921 68.5 (54.7–82.3) 70.75 (57.1–84.4) 68.3 (54.4–82.1)

Male 927 62.0 52.8 62.8

White 921 66.9 65.3 67.0

Systolic blood pressure, mm Hg 927 139.9 (110.8–169) 145.5 (115.8–175.2) 139.4 (110.4–168.3)

Diastolic blood pressure, mm Hg 927 80.1 (60.8–99.4) 82.1 (59.7–104.5) 79.9 (60.9–99)

Heart rate, beats/min 927 88 (65.2–110.8) 87 (68.3–105.7) 88 (64.9–111.2)

Past medical history

Acute myocardial infarction 927 27.5 29.2 27.4

Coronary artery disease 927 46.1 54.2 45.4

Prior PCI 927 22.5 26.4 22.2

Prior CABG 927 17.0 25.0 16.4

Arrhythmia 927 47.7 44.4 48.0

Hypertension 927 80.7 87.5 80.1

Hyperlipidemia 927 52.4 52.8 52.4

Diabetes* 927 43.6 58.3 42.3

Cerebrovascular accident 927 13.9 8.3 14.4

Peripheral arterial disease 927 3.2 2.8 3.3

COPD 927 26.1 23.6 26.3

Chronic kidney disease† 927 25.9 45.8 24.2

Anemia 927 22.4 29.2 21.9

Liver failure 927 2.8 5.6 2.6

Tobacco use 927 16.6 12.5 17.0

Cancer 927 14.1 13.9 14.2

Prior medications

Beta-blockers 927 70.2 73.6 69.9

ACE inhibitors 927 43.7 38.9 44.1

Angiotensin-receptor blockers 927 18.9 26.4 18.2

Diuretic agents 927 70.7 68.1 70.9

Antiarrhythmic agent 927 15.3 18.1 15.1

Digoxin 927 11.0 6.9 11.3

Laboratory studies

Sodium, mg/dl 921 139 [136–141] 139 [136–142] 139 [136–141]

Creatinine, mg/dl† 927 1.19 [0.94–1.6] 1.60 [1.02–2.46] 1.17 [0.93–1.55]

eGFR, ml/min/1.73 m2† 927 57.0 [40.4–77.8] 38.1 [24.8–66.1] 58.1 [42.4–79.2]

Hemoglobin, g/dl† 924 11.8 [10.1–13.2] 10.4 [8.7–12.4] 11.9 [10.3–13.3]

BUN, mg/dl† 918 23.5 [16.5–36.0] 33.7 [21.3–60.8] 23 [16–35]

BNP, ng/l 529 795 [337–1,486] 810 [501–1,915] 793 [328–1,479]

NT-proBNP, ng/l 316 3,446 [1,552–7,376] 4,814 [2,389–7,567] 3,221 [1,507–7,360]

Troponin T, ng/ml* 343 0.029 [0.015–0.056] 0.045 [0.024–0.080] 0.027 [0.014–0.051]

Troponin I, ng/ml 479 0.04 [0.02–0.07] 0.04 [0.025–0.115] 0.04 [0.02–0.066]

Values are mean (range), %, or median [interquartile range]. *p < 0.01. †p < 0.001.

ACE ¼ angiotensin-converting enzyme; AKI ¼ acute kidney injury; BNP ¼ B-type natriuretic peptide; BUN ¼ blood urea nitrogen; CABG ¼ coronary artery bypass graft;COPD ¼ chronic obstructive pulmonary disease; eGFR ¼ estimated glomerular filtration rate; NT-proBNP ¼ N-terminal pro-B-type natriuretic peptide; PCI ¼ percutaneouscoronary intervention.

Maisel et al. J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6

Plasma NGAL for AKI in AHF S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1

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values for subjects with and without the primaryoutcome are shown in Figure 1B.

For the primary outcome, the AUC, 80% sensi-tivity cutpoint, and 80% specificity cutpoint forpeak NGAL were 0.656 (95% CI: 0.589 to 0.723),141.0 ng/dl, and 350.0 ng/dl, respectively (Figure 2A).The AUC, 80% sensitivity cutpoint, and 80% speci-ficity cutpoint for first NGAL were 0.647 (95% CI:0.579 to 0.715), 104.4 ng/dl, and 274.9 ng/dl,respectively. In comparison, the AUC for the first

creatinine value was 0.652 (95% CI: 0.576 to 0.729).The combination of first creatinine and first NGALdid not enhance predictive ability (AUC: 0.66;95% CI: 0.586 to 0.733).

In multivariate logistic regression analysis,admission systolic blood pressure, pre-existingchronic kidney disease, admission hemoglobin, andadmission BUN retained significance in the model. Nointeraction was found by region (United States vs.Europe). Peak NGAL (p ¼ 0.038) was significant when

FIGURE 1 Primary Outcome Events and NGAL Values

1.00

0.98

0.96

0.94

0.92

0.90

0.88

0.86

0.840 24 48 72 96 120

50 100 200 500 1000

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ent-

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Sur

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NGAL (ng/mL)

Number at risk 927 817 654 524 396 259

Peak, Event

Peak, No Event

First, Event

First, No Event

A

B

(A) Kaplan-Meier curve with subjects at risk at the bottom. Approximately 86% of subjects

were free from the primary outcome at 5 days. (B) Peak and first NGAL values are displayed

in those with and without the primary outcome. NGAL values were log-transformed for

nonnormalized distribution. NGAL ¼ neutrophil gelatinase-associated lipocalin.

J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6 Maisel et al.S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1 Plasma NGAL for AKI in AHF

1425

added to the multivariate model, but the first NGAL(p ¼ 0.071) was not. Findings did not differ on thebasis of region. The addition of peak NGAL did notsignificantly improve the C statistic of the model(AUC increased from 0.707 to 0.711; p ¼ 0.70) for theprimary outcome.

For the individual outcome of WRF, the AUCs forpeak NGAL, first NGAL, first creatinine, and combinedfirst NGAL and creatinine were 0.637 (95% CI: 0.566 to0.708), 0.625 (95% CI: 0.554 to 0.695), 0.631 (95% CI:0.552 to 0.711), and 0.636 (95% CI: 0.559 to 0.713),respectively. For the individual outcome of RRT, theAUCs for peak NGAL, first NGAL, first creatinine, andcombined first NGAL and creatinine were 0.884(95% CI: 0.796 to 0.961), 0.902 (95% CI: 0.827 to0.976), 0.876 (95% CI: 0.754 to 0.998), and 0.914(95% CI: 0.827 to 1.00), respectively.SECONDARY OUTCOMES. A total of 235 adverse out-comes occurred in 144 subjects (Figure 3A). The dis-tribution of peak NGAL value for subjects with eachadverse outcome and those without adverse outcomesare shown in Figure 3B. The most common adverseoutcome was nephrology consultation (n ¼ 59). A sig-nificant number of subjects were admitted to theintensive care unit requiring inotropic/vasopressorsupport (n¼ 51) or ventilator support (n¼ 43). Twenty-nine subjects (3.1%) died during hospitalization.

The AUC, 80% sensitivity cutpoint, and 80%specificity cutpoint for peak NGAL for the compositesecondary outcome were 0.653 (95% CI: 0.601 to0.704), 124.6 ng/dl, and 331.6 ng/dl, respectively(Figure 3C). AUC, 80% sensitivity cutpoint, and 80%specificity cutpoint for the first NGAL were 0.691(95% CI: 0.643 to 0.740), 109.3 ng/dl, and 247.5 ng/dl,respectively. AUC for the first creatinine value was0.686 (95% CI: 0.634 to 0.738). The combination offirst creatinine and first NGAL had an AUC of 0.716(95% CI: 0.666 to 0.766). Because nephrologyconsultation was the most frequent adverse outcome,an analysis without this outcome was performed.AUCs for peak NGAL, first NGAL, and first creatininenotably decreased, and were 0.595 (95% CI: 0.534 to0.656), 0.645 (95% CI: 0.589 to 0.701), and 0.623(95% CI: 0.562 to 0.684), respectively.

The alternative definitions of WRF evaluatedincluded a sustained increase of creatinine $50% ofthe first creatinine value, a nonsustained increase ofcreatinine of $0.5 mg/dl or $50% of the first creati-nine value, a sustained increase of creatinine of $0.3mg/dl or $50% of the first creatinine value, and anonsustained increase of creatinine of $0.3 mg/dlor $50% of the first creatinine value, occurring in thefirst 5 days, and a nonsustained increase of creatinineof $0.3 mg/dl or $50% of the first creatinine value at

any time. For these definitions, there were 38 (4.1%),112 (12.1%), 128 (13.8%), 222 (23.9%), and 284 (30.6%)events, respectively. Using the first NGAL, AUCsranged from 0.488 to 0.625, with the highest AUC fora nonsustained increase in creatinine of $0.3 mg/dlor $50% of the first creatinine value at any time.Evaluating peak NGAL value only in subjects whosecreatinine rose by $0.3 mg/dl, AUCs ranged from0.593 to 0.606. Again, the highest AUC was inthose with a nonsustained increase in creatinine of$0.3 mg/dl or $50% of the first creatinine value at anytime. In subjects with an eGFR <60 ml/min/1.73 m2,there was no correlation between first NGAL andpercent decline in eGFR at discharge.

FIGURE 2 Receiver Operating Characteristic Curves for the Primary Outcome and Components

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itivi

ty

0.0 0.2 0.4 0.6 0.8 1.01 - Specificity

1.0

0.8

0.6

0.4

0.2

0.0

Sens

itivi

ty

0.0 0.2 0.4 0.6 0.8 1.01 - Specificity

Peak NGALFirst NGALFirst Creatinine

AUC0.6560.6470.652

95% CI0.589-0.7230.579-0.7150.576-0.729

Peak NGALFirst NGALFirst Creatinine

AUC0.6370.6250.631

95% CI0.566-0.7080.554-0.6950.552-0.711

Peak NGALFirst NGALFirst Creatinine

AUC0.8840.9020.876

95% CI0.796-0.9610.827-0.9760.754-0.998

A B

C

Peak NGAL First NGAL First Creatinine

Receiver operating characteristic curves for primary outcome (A), individual components of worsening renal function (B), and renal-

replacement therapy (C). Each plot contains receiver operating characteristic for peak NGAL, first NGAL, and first creatinine, as well as AUC with

95% CIs. AUC ¼ area under the curve; CI ¼ confidence interval; NGAL ¼ neutrophil gelatinase-associated lipocalin.

Maisel et al. J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6

Plasma NGAL for AKI in AHF S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1

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SUBGROUP ANALYSIS. Subjects were first stratifiedby eGFR greater or less than 60 ml/min/1.73 m2.Subsequently, they were stratified at 2 cutpoints ofthe first NGAL: NGAL greater or less than 150 ng/dl,and greater or less than 109.3 ng/dl, the latter beingthe 80% sensitivity cutpoint for the composite sec-ondary outcome. The percent of subjects who had anyadverse outcome, by the different subgroups andNGAL cutpoints, is shown in Figure 4A.

The occurrence of adverse events increased withincreasing NGAL and decreasing eGFR. Subjects with

eGFR $60 ml/min/1.73 m2 and NGAL less than thecutpoint had the fewest adverse outcomes, followedby those with eGFR <60 ml/min/1.73 m2 and NGALless than the cutpoint, then eGFR $60 ml/min/1.73 m2

and NGAL greater than the cutpoint, and, lastly, thosewith eGFR <60 ml/min/1.73 m2 and NGAL greater thanthe cutpoint, who had a marked increase in adverseoutcomes (Figures 4B and 4C). In subjects with aneGFR <60 ml/min/1.73 m2 and NGAL less than thecutpoint, a cutpoint of 150 ng/dl had 84.5% sensitivityand a 91.0% negative predictive value for excluding

FIGURE 3 Outcomes for In-Hospital Adverse Events

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.30 5 10 15 20 25 30

Time (Days)

Even

t-Fr

ee S

urvi

val

Vent+Ino (17)

Ino (51)

Vent (42)

RRT (18)

Neph Cons (59)

Severe WRF (18)

Death (29)

None (775)

50 100 200 500 1000Peak NGAL (ng/mL)

Numberat Risk 927 317 95 38 16 8 2

1.0

0.8

0.6

0.4

0.2

0.00.0 0.2 0.4 0.6 0.8 1.0

1 - Specificity

Sens

itivi

ty

Peak NGALFirst NGALFirst Creatinine

Peak NGALFirst NGALFirst Creatinine

AUC0.6530.6910.686

95% CI0.601-0.7040.643-0.7400.634-0.738

A B

C

(A) Kaplan-Meier curve for the combined secondary outcome with subjects at risk is at the bottom. Approximately 50% of subjects at risk for an adverse

event experienced an event during hospitalization. (B) Peak NGAL values (log-transformed) for the individual components of the combined secondary

outcome are shown. (C) Receiver operating characteristic curves for the combined secondary outcome with peak NGAL, first NGAL, and first creatinine AUC

and 95% CI. ino ¼ inotrope/vasopressor; Neph Con¼ nephrology consultation; RRT ¼ renal-replacement therapy; Vent¼ ventilator; WRF ¼worsening renal

function; other abbreviations as in Figure 2.

J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6 Maisel et al.S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1 Plasma NGAL for AKI in AHF

1427

the development of an adverse outcome, whereas acutpoint of 109.3 ng/dl had a 90.3% sensitivity and91.1% negative predictive value (Figures 4B and 4C).

DISCUSSION

WRF during hospitalization for AHF is associatedwith significant morbidity and mortality (2–4,6). Bothmechanistic and iatrogenic perturbations are causa-tive, including diuretic therapy, with its neurohor-monal activation and maladaptive effects on renalfunction (18,19). Although serum creatinine isconsidered the standard functional biomarker fordiagnosing WRF, it often does not increase until 1 to 3

days after injury has occurred, delaying diagnosticassessment and potential management. Thus, there isa clear unmet need for biomarkers to diagnose WRFearlier during the treatment of AHF, to potentiallyprevent or ameliorate progressive kidney damage anddysfunction.

AKINESIS is one of the largest multicenter inter-national trials exclusively evaluating the role of bio-markers for predicting the development of WRF orrenal dysfunction requiring RRT in patients present-ing with AHF treated with diuretic agents. More spe-cifically, in this report, we examined a commerciallyavailable biomarker, plasma NGAL. Although analysisdemonstrated a potential role for plasma NGAL in

FIGURE 4 Subgroup Analysis By Admission eGFR and First NGAL for In-Hospital Adverse Outcomes

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0%

Any

Adve

rse

Outc

ome

eGFR ≥60NGAL <150

eGFR <60NGAL <150

eGFR ≥60NGAL >150

eGFR <60NGAL >150

P<0.001

Adverse Events 7.4% 9.0% 11.7% 27.7%

Adverse Events by eGFR and NGAL 150

100.0%

90.0%

80.0%

70.0%

60.0%

50.0%

40.0%

30.0%

20.0%

10.0%

0.0%

Any

Adve

rse

Outc

ome

eGFR ≥60NGAL <109.3

eGFR <60NGAL <109.3

eGFR ≥60NGAL >109.3

eGFR <60NGAL >109.3

P<0.001

Adverse Events 7.0% 8.9% 11.7% 24.5%

Adverse Events by eGFR and NGAL 109.3

GFR≥60 (n=414) GFR<60 (n=492)

NGAL (ng/ml)

Any AdverseOutcome

None

NGAL (ng/ml)

Any AdverseOutcome

None

<150(n=320)

>150(n=94)

<150(n=178)

>150(n=314)

7.4% 11.7% 9.0% 27.7%

92.6% 88.3% 91.0% 72.3%

<109.3(n=243)

>109.3(n=171)

<109.3(n=112)

>109.3(n=380)

7.0% 11.7% 8.9% 24.5%

93.0% 88.3% 91.1% 75.5%

Predictive PerformanceeGFR<60 NGAL<150 mg/mL

Sensitivity

Specificity

Positive Predictive Value

Negative Predictive Value

84.5%

41.7%

27.7%

91.0%

(CI 76.0%-90.5%)

(CI 36.8%-46.7%)

(CI 23.0%-33.0%)

(CI 85.8%-94.5%)

Predictive PerformanceeGFR<60 NGAL<109.3 mg/mL

Sensitivity

Specificity

Positive Predictive Value

Negative Predictive Value

90.3%

26.2%

24.5%

91.1%

(CI 82.8%-94.9%)

(CI 22.1%-30.9%)

(CI 20.4%-29.1%)

(CI 84.2%-95.3%)

A

B

C

(A) Percent of subjects with an adverse event stratified by eGFR and the 2 different NGAL cutpoints. Incidence of adverse events by subgroups of eGFR (B)

and NGAL (C), presented by increasing prevalence of events, with their respective sensitivities, specificities, positive predictive values, and negative pre-

dictive values displayed beside them. eGFR ¼ estimated glomerular filtration rate; GFR ¼ glomerular filtration rate; other abbreviations as in Figure 2.

Maisel et al. J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6

Plasma NGAL for AKI in AHF S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1

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CENTRAL ILLUSTRATION Potential DiagnosticAlgorithm for Neutrophil Gelatinase-AssociatedLipocalin for Acute Kidney Injury

NGAL not helpful in predicting

acutekidneyinjury

NGAL only slightly

enhances prediction for adverse

events

Patients with an estimated

2

Is NGAL a helpful renal biomarker?

Is NGAL a helpful renal biomarker?

eGFR <60 ml/min/1.73 m2

Patient presenting with acute heart failure (AHF)

Intravenous diuretic agents being given

NGAL not helpful in predicting

acutekidneyinjury

NGAL may help predict risk (or lack

of risk)for adverse

events

Maisel, A.S. et al. J Am Coll Cardiol. 2016;68(13):1420–31.

In patients presenting with AHF treated with intravenous diuretic agents,

the results of AKINESIS do not support the use of NGAL in patients

with an eGFR $60 ml/min/1.73 m2. However, in patients with an

eGFR <60 ml/min/1.73 m2, NGAL may identify patients at low risk for

adverse events. The proposed algorithm is the result of subgroup analysis

and requires further study for confirmation. AHF ¼ acute heart failure;

AKINESIS ¼ Acute Kidney Injury N-gal Evaluation of Symptomatic heart

faIlure Study; eGFR ¼ estimated glomerular filtration rate;

NGAL ¼ neutrophil gelatinase-associated lipocalin.

J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6 Maisel et al.S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1 Plasma NGAL for AKI in AHF

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predicting adverse outcomes, this was driven bynephrology consultation, and NGAL could not predictthe development of WRF better than creatinine. Thereare several possible reasons why NGAL was notsignificant.

First, WRF may be caused by multiple factors,including AHF therapies; concurrent illness; or car-diorenal syndrome, a complex state consisting ofmultiple pathophysiologic processes (5,20). AKINESISwas designed to assess early development of WRFwith diuretic therapy; however, we could notadequately assess other possible etiologies of WRF, orif WRF developed later during the hospital course orbefore presentation. Other, unaccounted factors mayhave contributed to the rise in NGAL or the develop-ment of WRF. NGAL has been associated with car-diovascular disease and may be a marker of diseaseseverity (21). This association with disease severitymay be reflected in our clinical composite secondaryoutcome.

Second, the definition and understanding of whatconstitutes AKI is changing (22,23). Renal injury canoccur to varying degrees at different sites of thekidney and by multiple different mechanisms. WRFmay be manifested as renal damage, functionalchange (classically termed pre-renal AKI), or both(classically termed acute tubular necrosis), withbiomarkers of kidney damage or dysfunctiondetecting different types of renal injury (24). Itseems that NGAL may not detect renal injury re-flected by an increase in creatinine imposed bydiuretic therapy (7). In a prior small study, NGAL didnot change with the withdrawal and reinstitution ofdiuretic therapy (25).

Third, the rate of WRF in AKINESIS was low, at7.1%, compared with prior studies of NGAL, whichhave reported rates of 11.8% to 33.8%; however, amore liberal definition of a creatinine increase$0.3 mg/dl was used to define WRF in these studies(15,16). The low rate of WRF likely stems from ourmore stringent definition of WRF. Using an increaseof $0.3 mg/dl as a definition, 30.6% patients devel-oped WRF in our cohort. Even with this definition,the predictive value of NGAL was low. Notably,NGAL’s predictive ability improved with increasingseverity of kidney injury, as previously reported (26).

NGAL did have fair prognostic ability for adverse in-hospital outcomes (AUC: 0.691), although this wassimilar to creatinine (AUC: 0.686), and was driven bynephrology consultation, with a decrease in AUC withremoval of nephrology consultation (AUC: 0.645).Previous studies have shown NGAL’s association withadverse events (27–29). In the GALLANT study, plasmaNGAL at discharge was shown to predict 30-day

readmission for AHF and all-cause mortality, with anAUC of 0.73, which is similar to the AUC in AKINESIS(27). Furthermore, when grouping patients by eGFRand first NGAL values, we saw a stepwise increase inadverse events, as previously reported (28,30).

An exploratory finding was the low rate of adverseoutcomes in subjectswith an eGFR<60ml/min/1.73m2

and NGAL <150 ng/dl, suggesting a possible role forNGAL (Central Illustration). However, this is a post hocsubgroup analysis, and is only hypothesis-generating.Further study is needed to see if this is a specificpopulation for which NGAL can risk stratify.

Our study has multiple strengths. It is one of thelargest prospective cohorts specifically studying

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE:

Deterioration of renal function has prognostic impor-

tance in patients with AHF, but a rise in the serum

creatinine level is neither timely nor specific. Plasma

levels of NGAL increase in acute kidney injury, and are

associated with adverse outcomes in patients with

chronic HF, but were no better than creatinine levels

in predicting WRF from diuretic therapy or adverse in-

hospital events in patients with AHF.

TRANSLATIONAL OUTLOOK: Further studies are

needed to evaluate the value of biomarkers other than

NGAL for predicting worsening renal function during

diuretic therapy of patients with AHF.

Maisel et al. J A C C V O L . 6 8 , N O . 1 3 , 2 0 1 6

Plasma NGAL for AKI in AHF S E P T E M B E R 2 7 , 2 0 1 6 : 1 4 2 0 – 3 1

1430

biomarkers of WRF in AHF. It suggests that to fullyuse tubular biomarkers to predict WRF and itscomplications in AHF, we might need to considermore robust definitions of WRF, including largerchanges in creatinine over longer periods of timebefore improvement occurs, and the associationwith extraneous factors, such as continued use ofangiotensin-converting enzyme inhibitors or othersuperimposed inflammatory conditions. Our datasupport the notion of Damman and Testani (7) thatmuch of the creatinine elevation in AHFmay be termedpseudo-WRF.

STUDY LIMITATIONS. Samples were collected afterdiuretic therapy was administered, in part, to notdelay treatment, but this may have influenced NGALlevels. Samples were only collected early during thehospitalization, because the intent was to assess thedevelopment of early WRF. The first measuredcreatinine value was used as the baseline for assess-ment of WRF; however, this value may have alreadybeen elevated from a patient’s baseline creatinine.Patients may have been experiencing WRF onadmission not secondary to IV diuretic agents. Inaddition, creatinine measurements were not per-formed at a core laboratory and are subject tobetween-laboratory variability.

For outcomes, different definitions of WRF andadverse outcomes could have been used; however,analysis with alternative WRF definitions did notimprove NGAL diagnostic utility. Our endpoints wereon the basis of published reports at the time ofinception of AKINESIS, and additional exploratoryanalyses have been prompted by recent changes inthe understanding of WRF (22–24). Although in-hospital outcomes were closely monitored, eventswere not centrally validated. No interaction wasfound between NGAL and region; however, unmea-sured differences in delivery of care between theUnited States and Europe still could have influencedresults. Lastly, we lack post-discharge outcomes in

this analysis, which could have implications for theutility of NGAL.

CONCLUSIONS

AKINESIS is a novel, large, multicenter cohort studyexploring new biomarkers for AKI in AHF. AKINESISdoes not support the routine use of NGAL for earlierdetection of WRF or need for RRT in patientsadmitted with AHF and treated with diuretic agents.Also, NGAL overall was no more predictive of adverseoutcomes than creatinine. There was a signalthat a low NGAL value in patients with an eGFR<60 ml/min/1.73 m2 was sensitive for predicting alow risk of adverse outcomes; however, this is onlyhypothesis-generating.

REPRINT REQUESTS AND CORRESPONDENCE: Dr.Alan S. Maisel, VASDMC, Cardiology 111-A, 3350 LaJolla Village Drive, San Diego, California 92161.E-mail: amaisel@ucsd.edu.

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KEY WORDS biomarkers, cardiorenalsyndrome, diuretics, worsening renalfunction